Mini-Bioreactor System For 3-Dimensional Organoid Research And Development

This application claims the benefit of the filing date of U.S. Provisional Application 63/574,786, filed Apr. 4, 2024 and titled “Mini-Bioreactor System For 3-Dimensional Organoid Research And Development,” the entirety of which is incorporated herein by reference.

The present disclosure relates generally to systems, devices, and methods for cultivating, growing, developing, and/or researching organoids.

Organoids are 3-dimensional (3D) in vitro tissue cultures that are intended to mimic an organ or tissue in vivo. Organoids are generally used for research purposes because they strive to replicate the complexity of an organ on a miniaturized scale without needing to use a live organ or animal. For example, organoids can be used in drug development for drug discovery, drug screening, or precision medicine. Organoids can also be used to perform disease modeling. A wide variety of organs can be studies using organoids, including the brain, heart, lung, liver, and kidney.

However, growing and analyzing 3D tissue cultures is difficult. Organoids do not have structural integrity. Thus, if an organoid is grown on a flat surface, such as a petri dish, its round shape will become flattened due to the gravity. Additionally, neuro signal recording and stimulation is also difficult for organoids because positions of the implanted microelectrodes in the organoid change over time. Thus, the experimental data becomes inconsistent and unreliable. For these reasons, improved systems, devices, and methods for cultivating organoids is needed to perform reliable, long-term, and large-scale research using organoids.

One or more embodiments of the present disclosure may include a basket configured to support an organoid. The basket may include a bottom, a first side extending upward from the bottom, and a microelectrode array disposed on the bottom.

In some embodiments, the first side may be hemispherical. In some embodiments, the first side may be cylindrical. In some embodiments, the basket may also include a second side opposite the first side. In some embodiments, the basket may also include one or more perforations in at least said bottom or said first side. In some embodiments, the microelectrode array may include one or more electrodes configured to penetrate the organoid. In some embodiments, the microelectrode array may include one or more electrodes configured to contact a surface of the array.

One or more embodiments of the present disclosure may include a bioreactor system for organoids. The bioreactor system may include one or more single-unit bioreactors, a fluid handling system, and a processor. The one or more single-unit bioreactors may include a basket shaped to hold a three-dimensional organoid, a microelectrode array disposed within the basket, and one or more sensors. The fluid handling system may include a first holding chamber for a first fluid, and a dispensing tip coupled to the first holding chamber and configured to deliver the first fluid to at least one of the one or more single-unit bioreactors. The processor may be in communication with the microelectrode array and the one or more sensors and may be configured to receive a first plurality of electrical signals from the one or more sensors.

In some embodiments, the processor may be further configured to receive a second plurality of electrical signals from the microelectrode array. In some embodiments, the processor may be further configured to send a third plurality of electrical signals to the microelectrode array. In some embodiments, the processor may be further in communication with the fluid handling system. In some embodiments, the processor may be further configured to control the fluid handling system to dispense a particular amount of the first fluid into a first single-unit bioreactor of the one or more single-unit bioreactors. In some embodiments, the fluid handling system may further include a second holding chamber for holding a second fluid and the dispensing tip may be further configured to deliver the second fluid to at least one of the one or more single-unit bioreactors.

Additional aspects, features, and advantages of the present disclosure will become apparent from the following detailed description.

These figures will be better understood by reference to the following Detailed Description.

For the purpose of promoting an understanding of the principles of the present disclosure, reference will now be made to the implementations illustrated in the drawings and specific language will be used to describe them. It will nevertheless be understood that no limitation of the scope of the disclosure is intended. Any alterations and further modifications to the described devices, instruments, methods, and any further application of the principles of the present disclosure are fully contemplated as would normally occur to one skilled in the art to which the disclosure relates. In addition, this disclosure describes some elements or features in detail with respect to one or more implementations or figures, when those same elements or features appear in subsequent figures, without such a high level of detail. It is fully contemplated that the features, components, and/or steps described with respect to one or more implementations or figures may be combined with the features, components, and/or steps described with respect to other implementations or figures of the present disclosure. For simplicity, in some instances the same or similar reference numbers are used throughout the drawings to refer to the same or like parts.

When growing and/or researching organoids, it may be important to estimate the cellular and acellular state of the organoid across its entire volume without needing to label or damage the organoid. An organoid may include a necrotic core with a low number (i.e. percentage) of viable cells surrounded by an outer layer having a high number (i.e. percentage) of viable cells. Generally, the outer layer is the target for analyzing the organoid because it has a relatively higher percentage of viable cells. Although the effects of the necrotic core on organoid research is not well-understood, reducing the size of the necrotic core and thereby increasing the number of viable cells in the organoid may better mirror the physiological environment. Thus, reducing the size of the necrotic core may yield improved, more accurate, experimental results.

The cellular and/or acellular state of the organoid may be estimated based on one or more properties of the organoid. For example, these properties may include one or more of cell diversities (e.g. by cell type) throughout the cell, cell fraction (which may be the percentage of the organoid's volume that is occupied by cells), cell types, spatial organization, or the pressure, state, or size of the inner necrotic core. In some embodiments, the organoid state can be used to estimate the similarity among a group of organoids such as, for example, similarities between different organoids in the mini-bioreactor system. This may be important because, when performing an experiment on multiple organoids, ensuring that each organoid is similar may improve the accuracy of the experiment by reducing variability among the tests.

However, current methods of growing and/or analyzing organoids may not support long-term growth or incubation of organoids and, thus, may not be able to perform accurate, long-term experiments on organoids. Moreover, current methods may use labeling which may damage the organoid and/or make it more difficult or inaccurate to analyze the organoid.

Recognizing and taking into account the importance and utility of a methodology and system that can provide the improvements described above, the present disclosure aims to provide methods and systems for improving the growth, incubation, development, and/or research of organoids.

One or more aspects of the present disclosure provide devices, systems, and/or methods for analyzing the organoid state and other properties of the organoid to improve research using organoids. One or more embodiments described herein may allow for long-term experiments to be performed on organoids without the use of labeling. Electrophysiological data and/or biomarker closed-loop feedback may be measured to monitor the growth and/or incubation of the organoid and minimize the size of the necrotic core.

In some embodiments, the present disclosure describes a bioreactor system for growing, cultivating, and/or developing 3-dimensional (3D) organoids. In some embodiments, the bioreactor system may be miniaturized (a “mini-bioreactor system”). The mini-bioreactor system may allow research to be performed using the organoids being grown, cultivated, and/or developed by the mini-bioreactor system.

The mini-bioreactor system may include three distinct features. First, the mini-bioreactor system creates and maintains in vivo environment, through closed-loop feedback, for organoids to stay healthy so that accurate and repeatable experiments can be performed over a long period of time. Second, this system can learn from experience and predict outcomes from inputs using Artificial Intelligence (AI). Third, the system can be monitored and interact remotely in real-time through the Internet of Things (IoT).

The mini-bioreactor system may include one or more single-unit bioreactors, each of which is configured to hold an organoid and perform experiments on that organoid. The single-unit bioreactor may include an organoid basket that is shaped to support and hold a 3D organoid while allowing nutrients to contact the organoid and waste to be removed from it. A micro-electrode array may be disposed on the basket such that it contacts the organoid. One or more electrodes or microelectrodes on the array may be configured to penetrate the organoid. The micro-electrode array may be capable of measuring one or more properties that can be used to estimate the organoid state.

illustrates a mini-bioreactor system, according to one or more embodiments of the present disclosure. The mini-bioreactor systemmay include four parts or sub-systems that communicate with each other. These sub-systems may include a bioreactor chamber, a fluid handling system(shown in), a computer system, and a graphical user interface (GUI).

illustrates a perspective view of a bioreactor chamber, according to one or more embodiments of the present disclosure.illustrates a cross-sectional view of the bioreactor chamberinalong the-line. The bioreactor chambermay include a multi-well platethat includes one or more individual wells(shown in e.g.,). The multi-well platemay be covered by a top or chamber enclosure. The bioreactor chambermay be insulated. For example, in some embodiments, the chamber enclosureof the bioreactor chambermay include a layer of insulationdisposed within it to prevent heat from entering or escaping the bioreactor chamber.

illustrates a multi-well plate, according to one or more embodiments of the present disclosure. Each individual wellof the multi-well platemay be connected to an electricity source. For example, each individual wellmay be connected to an electricity source via electrical lead lines or electrical wires. The individual wellsmay be connected to each other in series and/or in parallel. The individual wellsmay be connected to a processorthat sends electrical signals to and/or receives electrical signals from each individual well. The multi-well platemay include any suitable number of individual wells. For example, the multi-well platemay have a 5×5 array of individual wells, a 10×10 array of individual wells, a 50×50 array of individual wells, a 100×100 array of individual wells, or an 8×12 array of individual wells.

In some embodiments, the multi-well platemay include multiple processors(or microchips). In some embodiments, each processormay be communicatively coupled to a certain number of individual wells. For example, a single processormay be configured to communicate with 4 individual wells. However, the processormay be configured to communicate with any suitable number of wells, such as 1, 2, 3, 5, 6, 7, 8, 9, or 10 individual wells.

illustrate another embodiment of a multi-well plate, according to one or more embodiments of the present disclosure.illustrates an assembled view of the multi-well plateandillustrates an exploded view of the multi-well plate. In the illustrated embodiment, the multi-well plateis a 96-well plate having an 8×12 array of individual wells. The multi-well platein the illustrated embodiment includes four parts: a frame, bottomsfor each individual well, an electrical plate, and a well plate. The frameincludes holesfor fitting the bottomsof the individual wells. The bottomsmay include one or more electrical probes for contacting the organoid, as described in more detail below. The well plateincludes cylinders that form the sidesof the individual wellsthat couple to the bottomswithin the holesof the frame. The electrical plateis configured to fit around the individual wells. The electrical platemay include one or more electrical leads(shown in) for coupling each wellto one or more processor(shown in). The electrical leadsmay be electrically coupled to the probes in the bottomsof the multi-well plate. In some embodiments, the one or more processorsmay also be disposed on or within the electrical plate.

In some embodiments, the electrical plateand/or the bottomsof the wellsmay be formed of a thin-film polymer. For example, in some embodiments, the electrical plateand/or the bottomsmay be formed using polyimide thin film. In some embodiments, the thickness of the thin-film polymer may be less than or equal to 10 micrometers. The thin-film polymer may be optically transparent so that optical imaging can be used to image the organoids in the multi-well plate. In other words, the thin-film polymer may be transparent in an optical image of the multi-well plate. In some embodiments, the electrodes may be formed using a material that optimizes the signal-to-noise ration (SNR). For example, in some embodiments, the electrodes may be formed from platinum, iridium, or a conductive polymer.

In some embodiments, the number and size of the electrical leadsmay be minimized to decrease the percentage of surface area that is blocked by the tracesduring imaging. For example, in some embodiments, the electrical leadsmay have a diameter of approximately 3 micrometers. In some embodiments, there may be only one leadfor each individual well.

In some embodiments, the multi-well platecan be fabricated through commercial-grade microfabrication techniques that are known in the art to improve the repeatability, scalability, and cost-effectiveness of the multi-well plate.

Returning to, the mini-bioreactor chambermay include one or more single-unit bioreactors. Each single-unit bioreactormay be a closed-loop system that maintains a suitable environment for organoids to live. Thus, each single-unit bioreactormay support a single experiment. Each single-unit bioreactormay be disposed in an individual wellof the multi-well plate.

The single-unit bioreactorsmay include an organoid basketthat is shaped and sized to hold an individual organoid. The organoid basketmay be covered by an organoid enclosure or top. The organoid enclosuremay be insulated to prevent heat from entering or exiting the single-unit bioreactor.

In some embodiments, multiple single-unit bioreactorsmay be connected to each other through a local area network (e.g., the internet or Wi-Fi) so that multimodal experiments can be performed. In a multimodal experiment, organoids with different biological functions in different single-unit bioreactorsmay be able to communicate with each other.

In some embodiments, each single-unit bioreactormay include one or more sensors. Each sensor may be configured to measure a different parameter. For example, in some embodiments, each single-unit bioreactormay include one or more of an oxygen sensor, a carbon dioxide sensor, a pH sensor, a temperature sensor, a glucose sensor, a metabolites sensor, a neurotransmitter sensor, a neuro signals sensor, or electrochemical impedance spectroscopy (EIS).

The oxygen sensor, carbon dioxide sensor, pH sensor, temperature sensor, glucose sensor, metabolites sensor, and/or neurotransmitter sensor may be considered physical or chemical sensors. The oxygen sensor, carbon dioxide sensor, pH sensor, temperature sensor, and/or glucose sensor may detect critical parameters that are important for organoid's survival. The metabolite sensors and/or neurotransmitter sensors may detect organoid's metabolic activities, gene expression phenotypes, physiological, and/or pathological responses.

The neuro signal sensor may be a microelectrode array (MEA)that is fabricated on a flexible and stretchable substrate. The MEA may have a teardrop configuration, which may be able to stretch to accommodate an organoid in different sizes and hang the organoid down into the media solution. The MEA may also be able to deliver an electric field to the organoid within the single-unit bioreactor.

In some embodiments, for each single-unit bioreactor, there may be two MEA designs. The first MEA design may be for surface recording and activation. The second MEA design may be for sub-surface recording and activation. The first MEA design may be disposed on the top of the organoid and the second MEA design may be disposed on the bottom of the organoid. In other embodiments, there is only one MEA design that contacts the bottom of the organoid. The EISmay be a tool that monitors the histology of organoids and their overall health in real-time. In some embodiment, the single-unit bioreactormay include a wave guide that directs optical light to the organoidso that the response can be analyzed.

The fluid handling systemmay be configured to deliver fluids to and/or remove fluids from each single-unit bioreactor. For example, the fluid handling systemmay deliver liquid media, which contains nutrients for the organoids to survive, and oxygen (O) gas into each single-unit bioreactorvia an injection port. In some embodiments, the Ogas may include other gases as well such as, for example, nitrogen gas. Moreover, the fluid handling systemmay remove metabolic waste from each single-unit bioreactorvia a waste drain. In some embodiments, the fluid handling systemmay pump the metabolic waste out from each single-unit bioreactor. In some embodiments, The influx of nutrients and Oand the discharging of the metabolic waste by the fluid handling systemis designed to mimic an in vivo environment. In some embodiments, the temperature of the liquid media may be maintained at 37° C. This may be designed to mimic body temperature, which is also 37° C.

illustrates a flow chart of the layers of the single-unit bioreactor, according to one or more embodiments. In some embodiments, the single-unit bioreactormay include three fluid layers. The first or top layermay receive liquid media and Othat is delivered to the single-unit bioreactor. In some embodiments, the single-unit bioreactormay include one injection port(as shown in) or, in other embodiments, may include multiple injections ports(as shown in). In this particular embodiment, the single-unit bioreactorincludes separate injection portsfor each fluid: a liquid media injection portand two Ogas injection ports. The top layermay include one liquid media injection portand one Ogas injection port.

The second or middle layerof the single-unit bioreactormay include the organoid. The middle layermay receive liquid media and Ofrom the top layerfor cultivating the organoid. A second Ogas injection portmay allow Ogas to be delivered to the middle layer.

The third or bottom layerof the single-unit bioreactormay receive and/or collect waste from the middle layerand/or top layer. The waste may be liquid or gaseous and may be produced by the organoid. The waste may also include excess fluid injected, such as excess liquid media and/or Ogas. A waste drainmay remove waste from the bottom layer.

illustrates a fluid handling systemand a multi-well plate, according to one or more embodiments of the present disclosure.illustrates a fluid handling systemincluding a robotic dispenserand a multi-well plate, according to one or more embodiments of the present disclosure. The fluid handling systemmay include a robotic dispenserthat delivers fluids into the bioreactor chambervia the injection port(shown in). The robotic dispenser may be controlled by the computer system, as described in more detail below. The robotic dispensermay be programmable for injecting an accurate amount of a fluid into each single-unit bioreactor.

In some embodiments, the robotic dispenser may also be configured to deliver liquid media and/or Ogas to each single-unit bioreactor. In some embodiments, the robotic dispenser may also be configured to remove waste from each single unit bioreactor. In some embodiments, the robotic dispenser system may deliver drugs to the organoids in the single-unit bioreactorsfor drug discovery and drug screening applications.

The fluid handling systemmay include one or more holding chambersfor holding one or more fluids for injecting into the bioreactor chamber. For example, there may be separate holding chambersfor liquid media, for Ogas, and/or for drugs. A syringe, pipet, or dispensing tipmay be connected to each of the holding chambers. The dispensing tipmay dispense a known or controllable quantity of each fluid into a particular single-unit well. The dispensing tipmay be moveable across the top of the multi-well plateso that it can deliver one or more of the fluids in the holding chambersto each single-unit bioreactor. The dispensing tipmay be connected to a first conveyance systemand a second conveyance system. The first conveyance systemmay move the dispensing tipalong a first axis. The second conveyance systemmay move the dispensing tipalong a second axis. In some embodiments, the first axis and the second axis may be perpendicular. The computer systemmay control the robotic dispenserby controlling the conveyance systems,to control movement of the dispensing tipand/or controlling the dispensing tipto dispense a certain amount of a certain fluid into a certain single-unit bioreactor.

The computer systemof the mini-bioreactor systemmay include any suitable components. The computer systemmay include one or more processors including, for example, central processing units, multi-core processors, microprocessors, microcontrollers, digital signal processors, field programmable gate arrays (FPGAs), application specific integrated circuits (ASICs), graphics processing units (GPUs) and/or the like. The computing devicemay be implemented as a stand-alone subsystem, as a board added to a computing device, and/or as a virtual machine.

The computer systemmay also include a memory that may be used to store software executed by computing deviceand/or one or more data structures used during operation of computing device. The memory may include one or more types of machine-readable media. Some common forms of machine-readable media may include floppy disk, flexible disk, hard disk, magnetic tape, any other magnetic medium, CD-ROM, any other optical medium, punch cards, paper tape, any other physical medium with patterns of holes, RAM, PROM, EPROM, FLASH-EPROM, any other memory chip or cartridge, and/or any other medium from which a processor or computer is adapted to read.

The processor and/or the memory may be arranged in any suitable physical arrangement. In some embodiments, the processor and/or the memory are implemented on the same board, in the same package (e.g., system-in-package), on the same chip (e.g., system-on-chip), and/or the like. In some embodiments, the processor and/or the memory include distributed, virtualized, and/or containerized computing resources. Consistent with such embodiments, the processor and/or the memory may be located in one or more data centers and/or cloud computing facilities.

In some examples, the memory may include non-transitory, tangible, machine readable media that includes executable code that when run by one or more processors may cause the one or more processors to perform the methods described in further detail herein.

In some embodiments, the memory of the computer systemmay include executable instructions or programs for a spike detection model that can be executed by a processor of the computer system. The spike detection model may receive measurements indicating the detection of action potential by one or more sensorsgenerated at the organoids. Additionally, the memory of the computation systemmay include executable instructions or programs for impedance spectroscopy that can be executed by the processor of the computer system. The impedance spectroscopy instructions/programs may measure impedance of a single organoid.

Moreover, the computation systemmay include one or more artificial intelligence (AI) models. An AI model may be trained to predict physical, chemical, or electrical stimuli for manipulating gene expressions of one or more of the organoids in the bioreactor chamber. In some embodiments, an AI model may be trained to detect and analyze neuro signal patterns and generate biological responses based thereon. In some embodiments, an AI model may be trained to identify lead compounds for drug discovery applications. In some embodiments, an AI model may be trained to identify drug toxicity and adverse effects of potential drug targets. In some embodiments, an AI model may be trained to identify drug interactions. In some embodiments, an AI model may be trained to screen for effective drugs for a certain disease. In some embodiments, an AI model may be trained to create a personalized treatment protocol for one or more diseases.

The memory of the computer systemmay also include instructions or programs for an Internet of Things (IoT) model that can be executed by a processor of the computer system. The IoT model may provide global collaboration of various components of the mini-bioreactor systemat any point in time. The IoT model may provide real-time telemetry and closed-loop monitoring of one or more components of the mini-bioreactor system. The IoT model may observe data collected by one or more sensors in the bioreactor chamber. The IoT model may instruct the processor and/or memory computer systemto take immediate action based on the data.

In some embodiments, the IoT model may allow for interaction between organoids. For example, the IoT model may allow organoids in the system to communicate with each other through wireless communication such as the internet.

In some embodiments, the computer systemmay control each single-unit bioreactorso that experiments can be performed on the organoidstherein. For example, the computer systemmay control, via electrical signals, the microelectrode arrayto apply an electrical stimulus to the organoid. The computer systemmay then receive electrical signals from the microelectrode arrayand/or the sensors,. The electrical signals may represent data collected by the MEAand/or the sensors,. The computer systemmay analyze this data.